【作者】 毕影东；

【导师】 杨传平； 王柏臣；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 植物芽休眠是植物生长发育过程中经过长期演化而获得的一种对环境条件及季节性变化的生物学适应性。芽的休眠特性不仅对物种的保存、繁衍具有特殊的生物学意义,而且在农林生产中具有重要的经济意义。樟子松是我国北方半干旱风沙地区营造防风固沙林、农田防护林、牧场防护林、水土保持林和用材林的主要树种之一。但若不能正确掌握樟子松顶芽休眠与萌发生物学特性及萌发过程中的生理生化变化规律,尤其是对樟子松芽休眠解除机理的认识不足,往往会对引种、选种以及杂交育种等工作产生较大影响,严重制约了樟子松遗传改良的进程。植物芽休眠与解除是个复杂的生命过程,涉及到新陈代谢,激素调控,营养物质合成及转运,细胞信号转导,细胞分裂及分化等多方面。多年来,科研工作者对休眠进行了许多研究和探索,在种子休眠的研究方面取得了丰硕的成果,但在芽休眠方面起步较晚、研究较少,特别是在芽休眠机理、遗传学基础、休眠解除的调控等方面尚不清楚。随着新技术、新方法的出现,在芽休眠的研究上取得了较快的进展。蛋白质组学从一个生物体具有特定功能的器官、组织、细胞着手,在不同生理条件下研究表达蛋白质的变化,通过对蛋白质的表达模式和功能模式进行直接分析。它是后基因组学的一个重要部分,也是当前生物学领域研究的热点。本研究针对樟子松顶芽从冬季休眠到春季解除这一现象,观察从休眠到解除过程中樟子松芽的形态变化,从而确定对樟子松芽进行蛋白质组学研究的关键时期。采用蛋白质双向电泳和质谱联用的方法鉴定和分析樟子松顶芽休眠转换过程中表达丰度变化蛋白点的110个蛋白质点中,通过质谱鉴定了96个点,通过功能结构域搜索和相似性比较,对其中71个(74%)在数据库中已经有功能注释,25(26%)个为未知的或推测的蛋白质。利用KEGG PATHWAY对在数据库中已经有功能注释蛋白进行了GO分类进一步鉴定和分析其生物学功能。将这些蛋白质归属于7个主要的功能类群,包括：(1)细胞代谢和周期调控、(2)细胞构建、(3)蛋白质代谢、(4)胁迫反应、(5)木质素合成、(6)信号转导、转录调控、碳代谢等其它生物学功能,并且探讨了ABA诱导相关蛋白与胁迫相关蛋白质可能在芽休眠诱导和解除中发挥的作用,表明胁迫相关蛋白可能是樟子松顶芽休眠的诱导和解除过程的关键因子之一。鉴定出一个细胞周期调控因子Cell division cycle protein 48,具ATP活性并且参与质膜的重建,可能参与了芽休眠的诱导和解除调控的复杂过程。采用RACE-PCR技术继续对樟子松ACO基因进行克隆,得到1551bp的cDNA序列,编码516个氨基酸,计算分子量为56.58 kD,等电点为6.21。其氨基酸序列与其他物种的Aconitate hydratase (ACO)氨基酸序列具有较高的相似性。经生物信息学分析其结构和功能均具有顺乌头酸酶的特征。有研究也表明顺乌头酸酶与生长素和脱落酸的调控密切相关并且在植物发育过程中发挥作用,这预示,在植物生长发育过程中生长素、激素及顺乌头酸酶直接或间接的相互作用,从而在调控休眠与解除的过程中起一定作用。本研究初步揭示了参与樟子松芽休眠解除过程的蛋白质表达变化规律和生物学功能。这将有助于从整体水平上揭示林木芽休眠与解除的分子机理,对加快林木遗传育种的进程具有重要理论意义和应用价值。更多还原

【Abstract】 The bud dormancy in perennial plants, which formed during a long evolutional process, is a biological adaptability to the changes of environmental factors and different seasons. In particular, it is a characteristic feature of buds dormancy of many species which are of ecological interest in multiplication and economic interest in wood production. Pinus sylvestris L.is a major species for sand-fixation and major source of timber in North China for its high genetic adaptability to tolerate severe climatic conditions.Importantly, it will not provide a reference frame for future studies such as selection of elite trees, genetic breeding if we can not understand the complex molecular mechanisms of induce or break bud dormancy. Bud dormancy is a complex physiological process characterized by the cellular activities that involve metabolism, stress/defense response, gene regulation, signal transduction, and cell formation.Although many significant mileposts have been reached in our understanding of mechanisms of seed dormancy in the past years.it is not clear for mechanistic regulation of bud dormancy. Proteomics has become a basic method for the largescale analysis of proteins in many fields of plant biology. In combination with the availability of genome sequence data, proteomics has opened up enormous possibilities for identifying the total set of expressed proteins as well as expression changes both during growth and development.We focus on 4 critical stages of apical buds of Pinus sylvestris I.within periods of dormancy that are typically transition during bud development in a systems biology approach to unravel the underlying morphologic observation of apical bud development in Pinus. To identify key proteins related to bud dormancy and burst, a proteomic approach is being utilized. 2-DE was done using apical buds of 4 critical stages during the dormancy-to-growth transitions in Pinus sylvestris as a result of subsequent 110 differential expression protein spots(were detected in Coommassie-stained gels within the 4-7 pH) related to different stages.It allowed the confident identification of 96 out of the 110 protein spots subjected to LC MS/MS that was used to generate tryptic peptide masses that were submitted to Mascot for identification, Of the differentially expressed proteins,74%(71 protein points), a putative function was assigned based on similarity of sequences with previously characterized proteins,26%(25 protein points) did not hit any protein or were hypothetical proteins.Proteins were classified into 7 groups subject to KEGG PATHWAY analysis based on their biological process/pathway categories, respectively,(1)Cell metabolism;(2)cell wall formation;(3)protein metabolism;(4) stress/defense response;(5)lignin biosynthetic process;(6)other cellular functions that involve carbohydrate metabolism, transcriptional regulation, signal transduction. In the present study, dormancy and breaking of buds and the participation of the hormones ABA and stress-induced protein in this process were analyzed by means of proteomics.A cell division cycle protein 48 which may control bud development was identified.A putative germination-associated cDNA,aconitase (Psy-ACO)were cloned by means of RACE-PCR; The high identity match to available aconitase sequence,it has character of ACO by analysis amino acid polypeptide encoded by Psy-ACO.Taken together, these results suggest that aconitase plays a role in regulating the bud development.This study expands our understanding of the changes in protein expression associated with the dormancy-to-growth transitions in Pinus sylvestris and provides insights into the molecular mechanisms of induce or break bud dormancy. It is important in both theory and application to forest genetic breeding.更多还原